The present invention relates to methods of ex-vivo and in-vivo controlling the proliferation and differentiation of stem and progenitor cells. More specifically, the present invention relates to ex vivo and in vivo methods of promoting proliferation, yet restricting differentiation of stem and progenitor cells by treating the cells with transition metal chelates, copper chelates in particular. In another aspect, the present invention relates to a method of enriching a population of non-differentiated stem or progenitor cells present in a mixed population of cells cultured ex vivo by treating the cells with transition metal chelates or transition metal chelators. In yet another aspect, the present invention relates to ex-vivo expanded populations of stem and progenitor cells obtained by the methods of the present invention.
As used herein throughout, the phrase “transition metal chelator” refers to a transition metal ligand that has at least two atoms capable of coordinating with an indicated metal, so as to form a ring. A transition metal chelator of an indicated transition metal, is free of, i.e., not complexed with, an ion of the indicated transition metal and hence, the phrase “copper chelator”, for example, refers to a chelator of copper, which is free of, i.e., not complexed with, a copper ion.
As used herein throughout, the phrase “transition metal chelate” refers to a chelator of an indicated transition metal, as is defined hereinabove, which is complexed with an ion of the indicated transition metal and hence, the phrase “copper chelate”, for example, refers to a chelator of copper complexed with a copper ion.
As is well known in the art, one or more molecules are considered as transition metal chelators if the formation of a cyclic complex of the molecule(s) with an ion of the transition metal results in a “chelate effect”. The phrase “chelate effect” refers to the enhanced stability of a complexed system containing the chelate, as compared with the stability of a system that is as similar as possible but contains none or fewer rings. The parameters for evaluating the chelate effect of a chelate typically include the enthalpy and entropy changes (ΔH and ΔS), according the following equation:ΔG0=ΔH0−TΔS0=−RT ln βwhere β is the equilibrium constant of the chelate formation and hence represents the chelate effect.
Hence, transition metal chelates and copper chelates in particular refer to complexes that include copper ion and one or more copper chelator(s) complexed therewith, which are characterized by a large β value. Representative examples of copper chelators include polyamine molecules such as ethylene diamine and cyclam, which form copper chelates with enhanced chelate effect.
Normal production of blood cells (hematopoiesis) and of other cell types involves the processes of proliferation and differentiation which are tightly coupled. In most hematopoietic cells, following cell division, the daughter cells undergo a series of progressive changes that eventually culminate in fully differentiated (mature), functional blood cells, which in most part are devoid of or very restricted in proliferative potential. Similarly, for cells of other, non-hematopoietic origin, following cell division, the daughter cells undergo a series of progressive changes which eventually culminate in fully differentiated (mature) functional tissue, which in most part is composed of cells devoid of or severely restricted in proliferative potential. Thus, the process of differentiation limits, and eventually halts cell division. Only in a small minority of the cells in an organ, known as stem cells, cell division may result in progeny which are similar or identical to their parental cells. This type of cell division, known as self-renewal, is an inherent property of stem cells and helps to maintain a small pool of stem cells in their most undifferentiated state. Some stem cells lose their self-renewal capacity and following cell division differentiate into various types of lineage committed progenitors which finally give rise to mature cells. While the latter provide the functional capacity of the tissue, e.g., the blood cell system, the stem cells are responsible for the maintaining of tissue formation, e.g., hematopoiesis, throughout life, despite a continuous loss of the more differentiated cells through apoptosis (programmed cell death) and/or, e.g., for the blood system, active removal of aging mature cells by the reticuloendothelial system and/or other loses of cell mass. It will be appreciated that in one way or another these processes characterize all cell lineages of multicellular organisms, because replenishment of dead cells occurs during the life cycle of such organisms.
Normal hematopoiesis is coordinated by a variety of regulators which include glycoproteins such as the colony stimulating factors (CSF), as well as small molecules such as the retinoids. They regulate the survival (e.g., by inhibiting apoptosis), proliferation and differentiation of progenitor and precursor cells and the activation state of mature cells.
In acute leukemia, for example, there is a block in cell differentiation. As a result, the leukemic cells maintain their proliferative potential. Leukemic cells do not respond normally to the various regulators [37-42].
Thus, cells obtained from patients with acute myeloid leukemia develop in culture, in response to stimulation by colony stimulating factor (CSF), small colonies of undifferentiated cells, as compared to large colonies of granulocytes and macrophages, which develop following cloning normal hematopoietic cells.
Adult stem cells are typically very rare, whereby for most tissues the number of stem cells is 1 in a 1,000,000 cells. Hence, obtaining a large number of stem cells, especially human adult stem cells directly from a tissue of choice, is impractical.
Therefore, and as is further detailed below, expansion of the stem cell and other defined progenitor cells such as blood stem cells or lympho-hematopoietic progenitor cell subpopulations by ex-vivo culturing could have important clinical applications. Similarly, expansion of non-hematopoietic adult stem cell, such as stem cells isolated from organs such as liver, pancreas, kidney, lung, etc., by ex-vivo culturing could have important clinical applications, especially in view of recent findings showing that adult stem cells are capable of transdifferentiation, i.e., developing into cell lineages different from the lineages characterizing their tissue origin.
A variety of protocols have been suggested and experimented for expansion of such cell populations. The main experimental strategies employed include incubation of mononuclear cells with or without selection of CD34+ [8]; with different cocktails of early and late growth factors [17]; with or without serum [7]; in stationary cultures, rapid medium exchanged cultures [18] or under continuous perfusion (bioreactors) [6]; and with or without established stromal cell layer [19].
Although a significant expansion of intermediate and late progenitors was often obtained during 7-14 days ex-vivo cultures under these conditions, the magnitude of early hematopoietic (CD34+CD38−) stem cells with high proliferative potential, typically declined [6].
Thus, these cultures clearly do not result in true stem cell expansion, but rather in proliferation and differentiation of the stem cells into pre-progenitor cells, accompanied by depletion of the primitive stem cell pool.
In order to achieve maximal ex-vivo expansion of stem cells, the following conditions should be fulfilled: (i) differentiation should be reversibly inhibited or delayed; and (ii) self-renewal should be maximally prolonged.
For some applications, following cell expansion, it is important to have methods to induce differentiation of the expanded cell population, so as to convert the expanded cell population to mature functional cells or tissue. In other applications, expanded undifferentiated stem cells can be used in their undifferentiated state to augment stem cell deficiency or be used in in vivo transdifferentiation applications.
International Patent Application Serial Nos. PCT/IL99/00444 and PCT/US99/02664, U.S. patent application Ser. Nos. 09/986,897 09/988,127, and Peled et al. (Brit. J. Haematol. 116:655, 2002) teach that certain trace-element chelators, copper chelators in particular, can inhibit differentiation of stem and progenitor cells, thereby prolonging cell proliferation and expansion ex-vivo. It is further disclosed that elevation of cellular copper content accelerates stem or progenitor cells differentiation. It was thus postulated that cellular copper is involved in the modulation of stem or progenitor cell self-renewal, proliferation and differentiation, whereas, increasing cellular copper content accelerates differentiation of stem or progenitor cells, while decreasing of cellular copper content inhibits differentiation of stem or progenitor cells.
The mechanisms controlling the rate of self renewal versus differentiation in adult stem cells are not fully understood, nevertheless, as a response to harsh medical treatments, such as chemotherapy and/or radiotherapy, stem cell depletion below an adequate level, results in rapid loss of tissue due to impaired tissue regeneration. Under such circumstances, stem cell transplantation and/or treatment for augmenting in vivo stem cell renewal are advised [43-47].
There is thus an identified need for and it would be advantageous to have ex-vivo and in vivo methods useful in expanding stem and progenitor cells of various cell lineages.